Dear scientists,
I have encountered a problem where my bacteria (Staphylococcus Epidermidis) grow perfectly fine in liquid media (Tryptic soy broth) but not on agar plates (freshly prepared TSB plates). For the TSB plates that did grow bacteria, often only one small corner grew but not other parts although I streaked all over the plate using a glycerol stock (image1). I also plated the diluted bacteria solution from a liquid culture (OD was about 0.06) on the plates yet nothing grew (image 2). When I used the bacteria that did grow on the plates to streak another agar plate (TSB), they did grow but the middle of the plate didn’t grow anything (image 5). However, when I used a 6 month old LB agar plate for streaking, the bacteria grew perfectly fine (image 3). In addition, I also used an E. Coli liquid culture to streak a TSB plate, and they grew perfectly fine (image 4). I don't understand why my bacteria have problem growing on TSB agar plates but can grow in liquid TSB media. TSB media is recommended by ATCC for the growth of S. Epidermidis. This problem has halted all of my CFU experiments as the bacteria don't grow on agar plates. Would you be able to give me any suggestions why this is happening? Your time and help are strongly appreciated!
The simplest explanation would be that someone made an error for this batch of TSB plates, either one ingredient was at the wrong concentration or maybe they accidentally added an antibiotic. So before panicking I would make a new batch of plates and just try again.
Also double check that you have the same recipe for TSB broth and plates (other than the addition of agar).
Lastly, if LB is working for you then maybe it would be easiest to just go with LB plates.
@Michael J. Benedik
Thank you so much for answering my question! The recipe for TSB media is 15 g/ 500 ml of water (suggested by sigma), and that for TSB plates is 6 g of TSB + 3 g of agarose in 200 ml of water. The agar concentration is 1.5%, and the TSB concentration is the same for TSB broth and plates. The only thing I am worried about when making the plates is that I didn't put the agar flask into a 56 oC water bath after autoclaving to lower the temperature. But I will definitely make a new batch of TSB plates and try again!